1. Field of the Invention
The present invention relates to an insecticidal protein. More particularly, the invention relates to cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in certain cyanobacterium and resulting dipteracidal activity.
2. Description of Related Art
Mosquitoes and blackflies are representative of the order Diptera which are pests that have plagued humans and animals for generations. Mosquitoes are the major vectors for a number of human and animal diseases, including malaria, yellow fever, viral encephalitis, dengue fever and filariasis.
Various pesticides have been developed with the hope of controlling Diptera. Unfortunately, many such pesticides are associated with drawbacks which make the search for new pesticides a desirable one. For example, many pesticides exert a broad spectrum of activity and, as a consequence, destroy many non-target organisms which may be beneficial to the environment. Moreover, pesticides are frequently toxic to humans and animals and create environmental hazards in the locus of application.
Biopesticides are a class of naturally occurring pesticides frequently derived from unicellular or multicellular organisms which have developed natural defense against other organisms. The group of microorganisms pathogenic for insects is varied and diverse. The gram-positive soil bacterium Bacillus thuringiensis subsp. israelensis is one of many B. thuringiensis strains able to produce insecticidal proteins. These proteins, expressed during the sporulation cycle of the bacterium, assemble into parasporal crystalline inclusion bodies. The parasporal crystal produced by B. thuringiensis subsp. israelensis is toxic when ingested by the larvae of Diptera, including mosquitoes and black flies. Upon ingestion, crystal proteins are solubilized in the larval midgut and act in a manner not yet clearly defined to disrupt the epithelium of the larval midgut region. Swelling and/or lysis of the epithelial cells is followed by larval death from starvation.
In the larval stage, many Diptera are filter feeders that feed primarily on microscopic algae and bacteria. In view of these facts and because of the very selective nature of the toxic effects of the B. thuringiensis subsp. israelensis crystal proteins, there has been interest in employing B. thuringiensis subsp. israelensis as an agent for Dipter control. There are, however, two major difficulties encountered in attempting to employ B. thuringiensis subsp. israelensis as both an effective and environmentally safe biolarvicide. First, one protein component of the B. thuringiensis subsp. israelensis crystal, the cytA gene product, has expressed hemolytic activity in the presence of cultured mammalalian cells. Second, sprayed B. thuringiensis subsp. israelensis spores demonstrate a limited field life as insecticides, i.e., B. thuringiensis strains are not natural competitors in the aquatic environments in which many Diptera breed, and the spores quickly sink out of the upper few decimeters of lakes and streams, where most larval feeding occurs.
In order to overcome both of these difficulties and to present feeding larvae with a continuous pesticide exposure, there has been interest in transferring individual genes coding for nonhemolytic mosquito-toxic B. thuringiensis subsp. israelensis proteins into bacterial species that could more successfully compete in aquatic larval feeding locales.
Several major polypeptides have been identified as components of the B. thuringiensis subsp. israelensis insecticidal endotoxin, i.e., polypeptides with molecular mass of 27, 72, and 130 kDa, respectively. The 27 kDa protein is known to be hemolytic, but it is unclear whether the 27 kDa protein is mosquitocidal per se. Although none of the other crystal proteins expresses hemolytic activity, each has been found to exert larvicidal effects on at least some mosquito species.
The gene encoding the 130 kDa protein has been cloned and inserted into both E. coli and a cyanobacterium, Synechocystis 6803. See PCT International Pub No. WO 88/06631. Although it is reported therein that transformed Synechocystis 6803 expressed a fusion protein containing the 130 kDa protein, no data is supplied as to the effectiveness of such Synechocytis 6803 as a mosquitocidal.
The gene encoding the 130 kDa protein has also been cloned and inserted into another cyanobacterium, Agmenellum quadruplicatum PR-6. See Angsuthanasombat, et al., Appl. Environ. Microbiol. 55:2428-2430 (1989). Although when sonified, the transformed cyanobacterium was found to exert some mosquitocidal activity, the authors state that greater expression of undegraded 130-kDa protein will be necessary for effective control of tropical disease vectors. Other attempts have been made to create an effective dipteracidal cyanobacterium but with little or no success. See Chungjatupornchai, W., Current Microbiology, 21:283-288 (1990). One reason given therein is that the intracellular concentration of mosquidicidal protein is too low for toxic effects to be observed on larval feeding. Moreover, analysis of gel-fractionated cyanobacterial extracts reveals degradation of the expressed protein.
It is known that in translation and expression of proteins preferred codon usage may vary from species to species, i.e., certain species do not translate certain codons which other species routinely translate in making polypeptides. Thus, expression of an exogenous gene introduced from one species to another may be limited by the difference in codon usage observed between the original host of the gene and its new host. For example, preferred codons from Agmenellum quadruplicatum PR-6 are different from those of E. coli and/or Bacillus. See De Lorimer et al., Proc. Nat. Acad. Sci. USA, Vol. 81, pp. 7946-7950 (1984). Isoleucine encoding AUA codons are not used, or undergo de minimis translation in Agmenellum quadruplicatum PR-6. In the one known case of de minimus AUA translation in PR-6, there is no evidence that the protein is actually expressed by the organism. In Synechococcus elongatus analysis of the twenty eight known genes indicates no translation of AUA codons. Similarly, analysis of each of the four known genes of Synechococcus sp. WH7803, Synechococcus sp. WH8103 and Synechococcus vulcanus, respectively, indicates no translation of AUA codons. In Synechococcus sp. PCC 7942, analysis of the sixty-six known genes indicates de minimus translation of AUA codons, i.e., only four AUA codons out of nearly 1300 other isoleucine encoding codons. In Synechococcus sp. WH8020, one AUA codon is seen in the four known genes which contain thirty-eight other isoleucine encoding proteins. Therefore, it can reasonably be predicted that exogenous genes containing AUA codons could experience translation and expression problems in cyanobacteria. Indeed, failure of the above-described attempts to create an effective dipteracidal may be explained by differences in codon usage between Bacillus and the transformed cyanobacteria.
Thus, a shortcoming in the art has been failure to achieve sufficient expression of intact larvicidal protein in live cyanobacteria to kill Diptera larvae. The present invention overcomes this shortcoming and others and provides a transformant that is lethal to Diptera larvae.